In general, electrophoresis (EP) is a method of separating chemical substances (such as mixtures of proteins) by passing a mixture through a separation medium. An externally-applied voltage is used to drive the different molecules in the mixture through the separation medium at varying rates, usually in a convection-stabilized path to avoid channeling. Electrophoresis is described in various texts such as Electrophoresis: Theory, Techniques, and Biochemical and Clinical Applications by A. T. Andrews (Oxford Univ. Press, 1986) and in various patents classified in Class 204, subclasses 180-190, such as U.S. Pat. Nos. 4,292,154 (Ambler 1981) and 4,579,783 (Ogawa 1986).
In one common type of electrophoresis, the molecules of interest continue to move as long as voltage is applied to the separation medium. To prevent these molecules from being driven all the way through the gel, paper, or other separation medium, a marker compound such as bromphenol blue is usually added to the fluid being processed. When the marker has travelled a certain distance, the voltage is turned off and the separation process stops. The desired molecules, which have collected in relatively small zones, may be removed from the separation medium, or they may be visualized in the matrix by any of a variety of staining techniques.
A variant of electrophoresis, usually referred to as isoelectric focusing (IEF) or electrofocusing, involves passing a mixture through a separation medium which contains, or which may be made to contain, a pH gradient or other pH function. The device or gel has a relatively low pH at one end, while at the other end it has a higher pH. IEF is discussed in various texts such as Isoelectric Focusing by P. G. Righetti and J. W. Drysdale (North Holland Publ., Amsterdam, and American Elsevier Publ., New York, 1976).
As commonly used, the term "gradient" implies that there are no abrupt boundaries in a function which is changing. Under that definition, a graph of a pH gradient in an IEF device would be shown as a smooth curve with no sharp points. By contrast, the term "function" is broader; it includes gradients, but it also includes transitions which may contain abrupt changes at the boundary between two zones. It is possible to create pH functions with sharp boundary changes in IEF devices by various processes, such as (1) partially filling an IEF device with a certain substance, creating a gradient within that zone if desired; (2) halting the flow of liquid to the IEF device while the concentration of acid or other compound is increased or decreased to a desired level; and (3) adding more fluid to the IEF device. That will create two or more zones with widely varying pH levels Similarly, any other compound can be added, such as a different monomer which will form a different type of gel. The most commonly used gels are agarose and polyacrylamide.
The charge on a protein or other amphoteric molecule depends on the pH of the ambient solution. At the isoelectric point (pI) for a certain molecule, the net charge on that molecule is zero. At pH's above the pI, the molecule has a negative charge, while at pH's below the pI the molecule has a positive charge. Each different "ampholyte" (amphoteric electrolyte) has a characteristic isoelectric point.
When a mixture of ampholytes is electrophoresed in an IEF system, an anode (positively charged) is placed at the acidic end of the system, and a cathode (negatively charged) is placed at the alkaline end. Each ampholyte which has a net positive charge under the acidic conditions near the anode will be driven away from the anode. As it moves through the IEF system, it will enter zones having less acidity, and its positive charge will decrease. Each ampholyte will stop moving when it reaches its particular isoelectric point, since it no longer has any net charge at that particular pH. This effectively separates the ampholytes, since they have differtent pI's. The isolated molecules of interest can be removed from the IEF device by various means, or they can be stained or otherwise characterized.
Some types of IEF systems generate pH gradients by means of "carrier ampholytes." These are synthetic ampholytes which often have a significant amount of buffering capacity. When placed in an IEF device, each carrier ampholyte will seek its own isoelectric point. Because of their buffering capacity, many carrier ampholytes will establish a pH plateau rather than a single point. By using a proper mixture of carrier ampholytes, it is possible to generate a relatively smooth pH gradient for a limited period of time. Such mixtures are sold commercially under various trade names, such as Ampholine (sold by LKB-Produkter AB of Bromma, Sweden), Servalyt (sold by Serva Feinbiochemica of Heidelberg, FRG), and Pharmalyte (sold by Pharmacia Fine Chemicals AB, Uppsala, Sweden). The chemistry of ampholyte mixtures is discussed in various references, such as Righetti, supra, and in U.S. Pat. No. 3,485,736 (Vesterberg, 1969).
Carrier ampholytes suffer from various problems and limitations. They tend to be hydrolyzed by the electrodes, and they are relatively expensive. It has also been reported (Righetti, supra, at page 75) that commercially available carrier ampholytes do not perform as well as recycled carrier ampholytes. In addition, carrier ampholytes can interact with the proteins being separated, interfering with the separation process, and they can interfere with some visualization techniques. Carrier ampholytes also suffer from problems such as "cathodic drift" and the "plateau effect," which cause instability of the desired pH gradients.
U.S. Pat. No. 4,130,470 (Rosengren et al, 1978) discloses a method of generating an affixed pH gradient in an IEF device. That method involves the use of chargeable groups (such as carbonic, sulphonic, boric, or phosphonic acids, or amino groups) which are immobilized in the separation medium at a range of concentrations. That system also suffers from several limitations, including: (1) some proteins precipitate and form strong complexes with the charged group of the matrix, especially when aging monomer solutions are used to generate the pH gradient; (2) amino groups in the matrix adhere to untreated glass and other solid materials, making it difficult to remove gels from the devices after focusing, and making recovery of the sample difficult; (3) the reagents needed to prepare IEF media according to Rosengren et al are relatively expensive; (4) pre-manufactured gels normally must be dried and/or kept frozen for shipping and/or storage; and (5) gels made according to the method of Rosengren et al tend to suffer from swelling and endosmosis.
A different method of generating a pH gradient for use in IEF was developed by Troitsky et al, at the Crimean Medical Institute in the USSR. This method is described in Bull. Exp. Biol. Med. 75: 118-120 (1973; published in Russian) and in Biochim. Biophys. Acta 400: 24-31 (1975; published in English). This method is based on the fact that some borate compounds form acidic complexes with some organic molecules having polyhydroxyl groups (i.e., having more than one OH group; these are often called polyols). Borate-polyol complexes are discussed in, e.g., F. Cotton and G. Wilkinson, Advanced Inorganic Chemistry, 2nd ed., Volume 3 (John Wiley, New York, 1965). Based on that knowledge, Troitsky et al created IEF columns with buffer solutions containing borate compounds and gradients of certain polyhydroxyl compounds such as glycerol, mannitol, and sucrose. Those columns were used to perform IEF separation on several mixtures of proteins, such as human or rabbit hemoglobins.
Troitsky et al reported successful results with the specific protein mixtures they used, and they subsequently described several similar experiments using non-anchored polyols in Troitsky and Agitsky, Isoelectric Focusing of Proteins in Self-Organizing and Artificial pH Gradients, page 117; published in Russian by Naukova Dumke, Kiev, 1984. However, the systems they described suffer from several limitations. First, the acidic complexes formed by the borate have ionic charges, which causes them to move within the IEF device when voltage is applied. Second, because most polyhydroxyl compounds (such as glycerol and mannitol) are relatively dense, they were loaded into the bottoms of vertical columns. That tended to stabilize the placement of the polyols in the column while it remained vertical; however, it would have interfered with the use of horizontal IEF devices, which are preferred in some situations. In addition, it would be impractical to pre-manufacture and ship IEF devices having gradients generated according to the Troitsky method, or to store them for prolonged periods.
Various other reports of electrophoresis experiments describe the use of borate buffers and/or gels which contain polyhydroxyl compounds, although none use both ingredients in the manner described below. For example, Kozulic et al created gels containing gradients of poly-NAT, the common name for polymerized N-acryloyl-tris(hydroxymethyl)aminomethane. The purpose of generating those concentration gradients was to generate a gradient of pore sizes, so the gels could be used as molecular sieves in electrophoresis, to separate large proteins according to their size. Kozulic et al also used gels containing NAT in uniform concentrations, with no gradients, for IEF separation of proteins using carrier ampholytes. Both sets of experiments are described in M. Kozulic et al, Analytical Biochemistry 163: 506-512 (1987). In addition, Kozulic et al used gels having uniform concentrations of NAT to separate nucleic acids, using buffers that are commonly used for nucleic acid separation. One such buffer contain Tris, the common name for tris(hydroxymethyl)aminomethane, and borate. That borate buffer reportedly caused the gels to swell, producing unsatisfactory results. That work is described in B. Kozulic et al, Analytical Biochemistry 170: 478-484 (1988).
The object of this invention is to create EP and IEF systems and devices which: (1) can be manufactured using relatively inexpensive reagents; (2) are highly stable while in use, and do not suffer from cathodic drift or plateauing; (3) cause low levels of interaction and interference with proteins and other substances being separated; (4) can be created reliably and precisely by lab technicians with minimal training; (5) can be pre-manufactured in large numbers using automated equipment; (6) can be shipped and stored for prolonged periods without the need for drying or freezing; (7) can be easily removed from holding devices; and (8) can be used under widely varying conditions.